The overall theme of this project is the enzymology of the base excision repair of oxidative damage to DNA. The enzyme 5-hydroxymethyluracil-DNA glycosylase (hmdUDG) has been purified 10,000 fold from calf thymus and primary amino acid sequence will be obtained. Primers will be constructed based on the sequence and the cDNA isolated. This will be followed by genomic cloning to make a knock out mouse. There is evidence that there are several DNA repair enzymes directed against thymine glycol residues in DNA. We have devised a technique based on the structure of the enzyme substrate complexes of these enzymes to isolate them in essentially a one step fashion. They will then be sequenced, cloned and knock out mice constructed. Similar enzymes are present in S. cerevesiae and we will isolate them, obtain their sequence and make single and double deletion mutants to evaluate their in vivo role.